Reurink Janine, de Vrieze Erik, Li Catherina H Z, van Berkel Emma, Broekman Sanne, Aben Marco, Peters Theo, Oostrik Jaap, Neveling Kornelia, Venselaar Hanka, Ramos Mariana Guimarães, Gilissen Christian, Astuti Galuh D N, Galbany Jordi Corominas, van Lith-Verhoeven Janneke J C, Ockeloen Charlotte W, Haer-Wigman Lonneke, Hoyng Carel B, Cremers Frans P M, Kremer Hannie, Roosing Susanne, van Wijk Erwin
Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.
Donders Institute for Brain Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands.
NPJ Genom Med. 2022 Jun 7;7(1):37. doi: 10.1038/s41525-022-00306-z.
The USH2A variant c.2276 G > T (p.(Cys759Phe)) has been described by many authors as a frequent cause of autosomal recessive retinitis pigmentosa (arRP). However, this is in contrast with the description of two asymptomatic individuals homozygous for this variant. We therefore assessed pathogenicity of the USH2A c.2276 G > T variant using extensive genetic and functional analyses. Whole genome sequencing and optical genome mapping were performed for three arRP cases homozygous for USH2A c.2276 G > T to exclude alternative genetic causes. A minigene splice assay was designed to investigate the effect of c.2276 G > T on pre-mRNA splicing, in presence or absence of the nearby c.2256 T > C variant. Moreover, an ush2a zebrafish knock-in model mimicking human p.(Cys759Phe) was generated and characterized using functional and immunohistochemical analyses. Besides the homozygous c.2276 G > T USH2A variant, no alternative genetic causes were identified. Evaluation of the ush2a zebrafish model revealed strongly reduced levels of usherin expression at the photoreceptor periciliary membrane, increased levels of rhodopsin localization in the photoreceptor cell body and decreased electroretinogram (ERG) b-wave amplitudes compared to wildtype controls. In conclusion, we confirmed pathogenicity of USH2A c.2276 G > T (p.(Cys759Phe)). Consequently, cases homozygous for c.2276 G > T can now receive a definite genetic diagnosis and can be considered eligible for receiving future QR-421a-mediated exon 13 skipping therapy.
USH2A基因变异c.2276 G > T（p.(Cys759Phe)）被许多作者描述为常染色体隐性遗传性视网膜色素变性（arRP）的常见病因。然而，这与两名该变异纯合子无症状个体的描述相悖。因此，我们通过广泛的基因和功能分析评估了USH2A基因c.2276 G > T变异的致病性。对3例USH2A基因c.2276 G > T纯合子的arRP病例进行了全基因组测序和光学基因组图谱分析，以排除其他遗传病因。设计了一个小基因剪接试验，以研究c.2276 G > T在存在或不存在附近c.2256 T > C变异的情况下对前体mRNA剪接的影响。此外，构建了一个模拟人类p.(Cys759Phe)的ush2a斑马鱼敲入模型，并通过功能和免疫组织化学分析进行了表征。除了USH2A基因c.2276 G > T纯合变异外，未发现其他遗传病因。对ush2a斑马鱼模型的评估显示，与野生型对照相比，感光细胞纤毛周膜上usherin的表达水平大幅降低，感光细胞胞体中视紫红质的定位水平升高，视网膜电图（ERG）b波振幅降低。总之，我们证实了USH2A基因c.2276 G > T（p.(Cys759Phe)）的致病性。因此，c.2276 G > T纯合子病例现在可以得到明确的基因诊断，并可被认为有资格接受未来QR-421a介导的外显子13跳跃疗法。
